Faculty Opinions recommendation of Structural plasticity of SARS-CoV-2 3CL Mpro active site cavity revealed by room temperature X-ray crystallography

Abstract The COVID-19 disease caused by the SARS-CoV-2 Coronavirus has become a pandemic health crisis. An attractive target for antiviral inhibitors is the main protease 3CL Mpro due to its essential role in processing the polyproteins translated from viral RNA. Here we report the room temperature X-ray structure of unliganded SARS-CoV-2 3CL Mpro, revealing the resting structure of the active site and the conformation of the catalytic site cavity. Comparison with previously reported low-temperature ligand-free and inhibitor-bound structures suggest that the room temperature structure may provide more relevant information at physiological temperatures for aiding in molecular docking studies.

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Ensemble-based enzyme design can recapitulate the effects of laboratory directed evolution in silico

Nature Communications ◽

10.1038/s41467-020-18619-x ◽

2020 ◽

Vol 11 (1) ◽

Cited By ~ 4

Author(s):

Aron Broom ◽

Rojo V. Rakotoharisoa ◽

Michael C. Thompson ◽

Niayesh Zarifi ◽

Erin Nguyen ◽

...

Keyword(s):

Directed Evolution ◽

Protein Design ◽

Active Site ◽

Room Temperature ◽

De Novo ◽

Conformational Ensemble ◽

Enzyme Design ◽

X Ray ◽

Conformational Ensembles ◽

X Ray Crystallography

Abstract The creation of artificial enzymes is a key objective of computational protein design. Although de novo enzymes have been successfully designed, these exhibit low catalytic efficiencies, requiring directed evolution to improve activity. Here, we use room-temperature X-ray crystallography to study changes in the conformational ensemble during evolution of the designed Kemp eliminase HG3 (kcat/KM 146 M−1s−1). We observe that catalytic residues are increasingly rigidified, the active site becomes better pre-organized, and its entrance is widened. Based on these observations, we engineer HG4, an efficient biocatalyst (kcat/KM 103,000 M−1s−1) containing key first and second-shell mutations found during evolution. HG4 structures reveal that its active site is pre-organized and rigidified for efficient catalysis. Our results show how directed evolution circumvents challenges inherent to enzyme design by shifting conformational ensembles to favor catalytically-productive sub-states, and suggest improvements to the design methodology that incorporate ensemble modeling of crystallographic data.

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Evolution of an enzyme conformational ensemble guides design of an efficient biocatalyst

10.1101/2020.03.19.999235 ◽

2020 ◽

Cited By ~ 5

Author(s):

Aron Broom ◽

Rojo V. Rakotoharisoa ◽

Michael C. Thompson ◽

Niayesh Zarifi ◽

Erin Nguyen ◽

...

Keyword(s):

Directed Evolution ◽

Protein Design ◽

Active Site ◽

Room Temperature ◽

De Novo ◽

Conformational Ensemble ◽

Enzyme Design ◽

X Ray ◽

Conformational Ensembles ◽

X Ray Crystallography

AbstractThe creation of artificial enzymes is a key objective of computational protein design. Although de novo enzymes have been successfully designed, these exhibit low catalytic efficiencies, requiring directed evolution to improve activity. Here, we used room-temperature X-ray crystallography to study changes in the conformational ensemble during evolution of the designed Kemp eliminase HG3 (kcat/KM 160 M−1s−1). We observed that catalytic residues were increasingly rigidified, the active site became better pre-organized, and its entrance was widened. Based on these observations, we engineered HG4, an efficient biocatalyst (kcat/KM 120,000 M−1s−1) containing active-site mutations found during evolution but not distal ones. HG4 structures revealed that its active site was pre-organized and rigidified for efficient catalysis. Our results show how directed evolution circumvents challenges inherent to enzyme design by shifting conformational ensembles to favor catalytically-productive sub-states, and suggest improvements to the design methodology that incorporate ensemble modeling of crystallographic data.

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Synthesis, structural characterization and catalytic activity of chlororuthenium(II) complexes with substituted Schiff base/phosphine ancillary ligands

Zeitschrift für Naturforschung B ◽

10.1515/znb-2020-0110 ◽

2020 ◽

Vol 75 (9-10) ◽

pp. 851-857

Author(s):

Chong Chen ◽

Fule Wu ◽

Jiao Ji ◽

Ai-Quan Jia ◽

Qian-Feng Zhang

Keyword(s):

Schiff Base ◽

Catalytic Activity ◽

Structural Characterization ◽

Room Temperature ◽

Molecular Structures ◽

Cationic Complex ◽

Neutral Complex ◽

Ancillary Ligands ◽

X Ray ◽

X Ray Crystallography

AbstractTreatment of [(η6-p-cymene)RuCl2]2 with one equivalent of chlorodiphenylphosphine in tetrahydrofuran at reflux afforded a neutral complex [(η6-p-cymene)RuCl2(κ1-P-PPh2OH)] (1). Similarly, the reaction of [Ru(bpy)2Cl2·2H2O] (bpy = 2,2′-bipyridine) and chlorodiphenylphosphine in methanol gave a cationic complex [Ru(bpy)2Cl(κ1-P-PPh2OCH3)](PF6) (2), while treatment of [RuCl2(PPh3)3] with [2-(C5H4N)CH=N(CH2)2N(CH3)2] (L1) in tetrahydrofuran at room temperature afforded a ruthenium(II) complex [Ru(PPh3)Cl2(κ3-N,N,N-L1)] (3). Interaction of the chloro-bridged complex [Ru(CO)2Cl2]n with one equivalent of [Ph2P(o-C6H4)CH=N(CH2)2N(CH3)2] (L2) led to the isolation of [Ru(CO)Cl2(κ3-P,N,N-L2)] (4). The molecular structures of the ruthenium(II) complexes 1–4 have been determined by single-crystal X-ray crystallography. The properties of the ruthenium(II) complex 4 as a hydrogenation catalyst for acetophenone were also tested.

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Binding of Phosphate and pyrophosphate ions at the active site of human angiogenin as revealed by X-ray crystallography

Protein Science ◽

10.1110/ps.13601 ◽

2001 ◽

Vol 10 (8) ◽

pp. 1669-1676 ◽

Cited By ~ 14

Author(s):

Demetres D. Leonidas ◽

Gayatri B. Chavali ◽

Anwar M. Jardine ◽

Songlin Li ◽

Robert Shapiro ◽

...

Keyword(s):

Active Site ◽

X Ray ◽

X Ray Crystallography

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Recent insights into lytic polysaccharide monooxygenases (LPMOs)

Biochemical Society Transactions ◽

10.1042/bst20170549 ◽

2018 ◽

Vol 46 (6) ◽

pp. 1431-1447 ◽

Cited By ~ 39

Author(s):

Tobias Tandrup ◽

Kristian E. H. Frandsen ◽

Katja S. Johansen ◽

Jean-Guy Berrin ◽

Leila Lo Leggio

Keyword(s):

Active Site ◽

Room Temperature ◽

Experimental Studies ◽

Binding Mode ◽

Structural Features ◽

Oxygen Species ◽

Animal Kingdom ◽

X Ray ◽

Lytic Polysaccharide Monooxygenases ◽

Polysaccharide Monooxygenases

Lytic polysaccharide monooxygenases (LPMOs) are copper enzymes discovered within the last 10 years. By degrading recalcitrant substrates oxidatively, these enzymes are major contributors to the recycling of carbon in nature and are being used in the biorefinery industry. Recently, two new families of LPMOs have been defined and structurally characterized, AA14 and AA15, sharing many of previously found structural features. However, unlike most LPMOs to date, AA14 degrades xylan in the context of complex substrates, while AA15 is particularly interesting because they expand the presence of LPMOs from the predominantly microbial to the animal kingdom. The first two neutron crystallography structures have been determined, which, together with high-resolution room temperature X-ray structures, have putatively identified oxygen species at or near the active site of LPMOs. Many recent computational and experimental studies have also investigated the mechanism of action and substrate-binding mode of LPMOs. Perhaps, the most significant recent advance is the increasing structural and biochemical evidence, suggesting that LPMOs follow different mechanistic pathways with different substrates, co-substrates and reductants, by behaving as monooxygenases or peroxygenases with molecular oxygen or hydrogen peroxide as a co-substrate, respectively.

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Silylated gallium and indium chalcogenide ring systems as potential precursors to ME (E=O, S) materials

Open Chemistry ◽

10.2478/s11532-013-0255-y ◽

2013 ◽

Vol 11 (7) ◽

pp. 1225-1238

Author(s):

Iliana Medina-Ramírez ◽

Cynthia Floyd ◽

Joel Mague ◽

Mark Fink

Keyword(s):

Thermal Decomposition ◽

Room Temperature ◽

Membered Ring ◽

Molecular Structures ◽

Nmr Studies ◽

X Ray ◽

X Ray Crystallography ◽

High Yields ◽

Vt Nmr ◽

State 1

AbstractThe reaction of R3M (M=Ga, In) with HESiR′3 (E=O, S; R′3=Ph3, iPr3, Et3, tBuMe2) leads to the formation of (Me2GaOSiPh3)2(1); (Me2GaOSitBuMe2)2(2); (Me2GaOSiEt3)2(3); (Me2InOSiPh3)2(4); (Me2InOSitBuMe2)2(5); (Me2InOSiEt3)2(6); (Me2GaSSiPh3)2(7); (Et2GaSSiPh3)2(8); (Me2GaSSiiPr3)2(9); (Et2GaSSiiPr3)2(10); (Me2InSSiPh3)3(11); (Me2InSSiiPr3)n(12), in high yields at room temperature. The compounds have been characterized by multinuclear NMR and in most cases by X-ray crystallography. The molecular structures of (1), (4), (7) and (8) have been determined. Compounds (3), (6) and (10) are liquids at room temperature. In the solid state, (1), (4), (7) and (9) are dimers with central core of the dimer being composed of a M2E2 four-membered ring. VT-NMR studies of (7) show facile redistribution between four- and six-membered rings in solution. The thermal decomposition of (1)–(12) was examined by TGA and range from 200 to 350°C. Bulk pyrolysis of (1) and (2) led to the formation of Ga2O3; (4) and (5) In metal; (7)–(10) GaS and (11)–(12) InS powders, respectively.

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Synthesis, Molecular Docking Analysis, and Carbonic Anhydrase Inhibitory Evaluations of Benzenesulfonamide Derivatives Containing Thiazolidinone

Molecules ◽

10.3390/molecules24132418 ◽

2019 ◽

Vol 24 (13) ◽

pp. 2418

Author(s):

Zuo-Peng Zhang ◽

Ze-Fa Yin ◽

Jia-Yue Li ◽

Zhi-Peng Wang ◽

Qian-Jie Wu ◽

...

Keyword(s):

Carbonic Anhydrase ◽

Active Site ◽

Docking Studies ◽

Active Site Residues ◽

Single Crystal Diffraction ◽

Docking Analysis ◽

X Ray ◽

Active Site Cavity ◽

Molecular Docking Analysis ◽

Positive Controls

To find novel human carbonic anhydrase (hCA) inhibitors, we synthesized thirteen compounds by combining thiazolidinone with benzenesulfonamide. The result of the X-ray single-crystal diffraction experiment confirmed the configuration of this class of compounds. The enzyme inhibition assays against hCA II and IX showed desirable potency profiles, as effective as the positive controls. The docking studies revealed that compounds (2) and (7) efficiently bound in the active site cavity of hCA IX by forming sufficient interactions with active site residues. The fragment of thiazolidinone played an important role in the binding of the molecules to the active site.

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